The present invention is within the field of early cancer prediction. More closely, the invention relates to a method and kit for predicting virus-associated cervical cancer in a human subject.
Infection by certain subtypes of human papillomavirus (HPV), in particular HPV 16 and HPV 18, has long been recognized as a major risk factor for cervical cancer and about 95% of cancer biopsies contain HPV DNA. While infection with HPV is common in young women in the ages 16-24, only less than 1% of women with oncogenic HPV smears develop cervical cancer. Therefore, known methods for testing for presence of HPV has a low predictive value.
Within prior art there are two main strategies to predict or diagnose cervical cancer. One strategy uses squamous intraepithelial lesion in cytology, or cervical dysplasia, as an indication of progression to cervical cancer. The other main strategy is to detect HPV nucleic acid in a patient sample either directly or following amplification of said nucleic acid, wherein the presence of HPV nucleic acid is taken as an indication of possible progression to cervical cancer.
Prior art has also concentrated on determining the exact HPV types, for example, U.S. Pat No. 5,580,970 describes amplification of low-oncogenic HPV genes, such as HPV 6 and 11, as an indication of lower risk of progression to serious cervical cancer, and high-oncogenic HPV genes as an indication of higher risk.
U.S. Pat. No. 5,795,722 describes amplification of (i) one or more control nucleic acids together with amplification of (ii) a conserved region of an analyte nucleic acid from a suspected pathogen in a patient sample as well as (iii) a region of the analyte nucleic acid used for sequencing. Following amplification, the sequencing region is captured from the amplification mixture and the remaining fragment mixture is separated electrophoretically to determine the relative amounts of conserved fragments and control fragments. Thereafter, the sequence of the sequencing region is determined as well as its pathogenic source.
Ylitalo et al. in J. Clin. Microbiol. 33: 1822-1828 describes detection of genital HPV types by amplification of the conserved E1 region of oncogenic HPV types. Quantification of viral load is not discussed.
Hitherto there is no known method of predicting cervical cancer several years before development of cancer.
The present invention provides a method and kit enabling early prediction of the clinical outcome of cervical cancer in situ (CCIS). According to the present invention, cervical cancer can be predicted in HPV positive women where the initial HPV positive smears were sampled several years before development of cancer.
In a first aspect, the present invention provides a method to predict the risk of progression to virus-associated cancer in a human subject, comprising
a) measuring the amount of a viral nucleic acid sequence or fragment thereof in a sample from the human subject;
b) measuring the amount of said sample;
c) relating the value from a) to the value from b) to obtain a value of relative viral load in said sample. In one embodiment the amount of said sample is measured by measuring a human nucleic acid sequence or fragment thereof,
d) using said value to estimate risk of progression to cervical cancer, with higher viral load meaning an increased risk of progression to cervical cancer.
The viral nucleic acid sequence is preferably nucleic acid from cervical cancer-associated genital human papillomavirus (HPV)-types, such as HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58. In a preferred embodiment, the viral nucleic acid is derived from the E1 gene, the E6 gene, the E7 gene, the L1 gene, or fragment(s) thereof. The human nucleic acid sequence is preferably genomic DNA from a nuclear gene.
In the method according to the invention, the measuring in step a) and b) is made by known methods for quantitative DNA or RNA analysis, such as polymerase chain reaction (PCR), in situ hybridization, NASBA, 3SR, hybrid capture etc.. Alternatively, the amount of the biological sample is measured by counting cells, cell staining, cell fluorescence, total DNA amount, using a technical device for obtaining identical amounts of biological sample, or using volume, weight or other means of normalizing for sample amount.
In a second aspect, the invention provides a kit for predicting HPV-associated cervical cancer by amplification of viral nucleic acid, comprising a) primers specific for a region of the conserved E1 gene of oncogenic HPV types; and b) primers specific for genomic nucleic acid in a sample from a human subject.
The primers in b) may be selected from any nuclear gene, preferably a single copy gene. The kit may also comprise DNA intercalating compounds, such as ethidium bromide and SYBR(copyright) Green.
In a preferred embodiment, the kit also comprises labelled HPV specific probes; and labelled probes specific for human genomic DNA. The labels may be fluorophores, radioactive isotopes, compounds for chemiluminiscent detection etc.. Examples of fluorophores are FAM (6-carboxyfluorescein), HEX (hexachloro-6-carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), TAMRA (6-carboxytetramethylrhodamine), ROX (6-carboxy-X-rhodamine), Cy5 (cyanine).
Preferred HPV probes are:
HPV 16 probe: 5xe2x80x2-ATAATCTCCTTTTTGCAGCTCTACTTTGTTTTT-3xe2x80x2
HPV 18 probe: 5xe2x80x2-CCGCCTTTTTGCCTTTTTCTGCCCACTAATT-3xe2x80x2
HPV 31 probe: 5xe2x80x2-TCTTCGTTTTGCTGTTTTACTGTTATTTTCTAT-3xe2x80x2
HPV 33 probe: 5xe2x80x2-TTTTCGTTTTCTGTATGTGCATTCTTTATTTTT-3xe2x80x2
HPV 35 probe: 5xe2x80x2-TCGTCGCTTTCGTGCTGTATTTTTATTTTCA-3xe2x80x2